Natural silk, e.g. spider silk, is an extraordinary material having a very high tensile strength in combination with a high extensibility. Due to these properties it has been tried for many years to prepare this material in larger amounts. Since it is not possible to use animals as e.g. spiders for this purpose, research is focussing on the investigation of methods in which the starting material for silk (e.g. spider silk) proteins is obtained recombinantly and then spun to a thread.
As raw materials there are used authentic silk proteins (recombinant proteins which are obtained by means of authentic sequences of the silk gene) and synthetic silk proteins (proteins based on synthetic genes, wherein their primary sequence widely corresponds to the natural sequences). The quality of an artificially produced thread is assumed to be defined by both the raw material used and the spinning method applied.
As in the natural spinning process, in the artificial spinning process, the silk proteins have to be transferred from a soluble form into an insoluble form, the structure of which shall be as identical to the authentic thread as possible. For this, the working group of Jelinski has developed a micro spinning apparatus which allowed spinning a few milligrams of silk proteins to silk threads with a length of several meters (Liivak et al., 1998). Silk of the spider Nephila clavipes dissolved in hexafluoroisopropanol was used as a starting material. The so dissolved protein was injected in a precipitation bath of acetone through a spinning nozzle. However, the threads obtained therewith were very refractory and did hardly show any structural similarity to natural silk threads (Seidel et al., 1998; Seidel et al, 2000). Primarily, by treating it with water and supplementary drawing the thread (post spin draw), both mechanical and structural parameters could be improved. However, the properties of natural silk have not been achieved (Seidel et al., 2000).
Another group developed a spinning technique, in which a methanol/water mixture was used as a precipitation bath. With this, a synthetic silk protein and a recombinant MaSp1 of the spider Nephila clavipes could be spun from an urea-containing solution. However, these were also refractory (Arcidiacono et al., 2002).
By using the same technique, recombinant ADF-3 being solved without chaotropic reagents could be spun to threads. Also in this case, the properties of the thread could be improved by post spin draw (Lazaris et al., 2002), though the tensile strength of natural threads has not been achieved. The companies Oxford Biomaterials (Oxford, Great Britain), Spin 'Tech GmbH (Ludwigsburg, Germany) and the Institut für Mikrotechnik Mainz GmbH (Mainz, Germany) have developed a method according to the state of knowledge of the inventors, with which silk proteins can be spun to threads by a microdialysis method or a similar method.
Additionally, there are successful trials to obtain threads from silk proteins by means of the so called electro-spinning (Prof. Frank Ko, Drexel University, Philadelphia, Pa., U.S.A.). However, there has not been disclosed anything about the mechanical properties of the so produced threads yet.
US 2003/0201560 relates to an apparatus for spinning threads from protein solutions. It is stated that the apparatus has a funnel-formed section through which the protein solution or “dope”, respectively, is passed, wherein this passage is at least partially consisting of a semipermeable and/or porous material.
WO 2005/017237 inter alia relates to an apparatus for assembling proteins. The apparatus has a tubular passage, the walls of which are partially permeable or porous. This has the advantage of monitoring the pH, the water content and the ion composition.
WO 2004/057069 relates to a method and an apparatus for preparing objects, especially also for spinning threads from spider silk proteins. This method essentially relates to the sol-gel transition of the protein solution which is for example achieved by adding potassium, preferably potassium fluoride. Furthermore, it is stated here that the apparatus used for performing the method has a semipermeably or porously formed “transition compartment”.
WO 2003/060099 refers to the preparation of spider silk fibres or bio-filaments, respectively. In the apparatus given, there is described an “extrusion unit” through which the spider silk protein solution is passed. WO 2003/060099 is especially directed to inserting the filaments in a coagulation bath after air contact.
Consequently, the previously used and publicated methods for spinning spider silk proteins mostly base on the injection of a protein solution in a precipitation bath. For stabilizing the soluble state of the proteins in the spinning solution, the precipitation bath usually contains chaotropic substances or organic solvents. For compensating the effect of these additives and inducing the protein assembly, lyotropic agents are accordingly added to the precipitation bath.